Molecular components of PAMP-triggered oxidative burst in plant immunity [Elektronische Ressource] / vorgelegt von Sophia Mersmann
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2009
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75
pages
Deutsch
Documents
2009
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01 janvier 2009
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Molecular components of PAMP-triggered oxidative burst in plant immunity
Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Sophia Mersmann aus München
Köln, September 2009
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Die vorliegende Arbeit wurde am Max-Planck-Institut für Züchtungsforschung in Köln in der Arbteilung für Molekulare Phytopathologie (Direktor: Prof. Dr. P. Schulze-Lefert) angefertigt.
Berichterstatter:
Prüfungsvorsitzender:
Tag der Disputation:
Prof. Dr. Paul Schulze-Lefert Prof. Dr. Martin Hülskamp Prof. Dr. Jaakko Kangasjärvi
Prof. Dr. Ulf-Ingo Fluegge
20. Oktober 2009
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Table of Contents Abbreviations ................................................................................................................................................7 Publications....................................................................................................................................................9 Summary......................................................................................................................................................10 Zusammenfassung........................................................................................................................................11 1 Introduction...............................................................................................................................................12 1.1 Reactive oxygen species (ROS).......................................................................................................12 1.2 ROS generating systems ..................................................................................................................12 1.3 ROS signaling .................................................................................................................................14 1.4 ROS-mediated processes ................................................................................................................15 1.5 Active plant immune responses........................................................................................................16 1.6 Receptors mediating PAMP-triggered defence responses ..............................................................17 1.7 PAMP mediated defence responses..................................................................................................18 1.8 Aim of thesis.....................................................................................................................................19 2 Material and Methods...............................................................................................................................20 2.1 Material............................................................................................................................................20 2.1.1 Plant Material..........................................................................................................................20 2.1.2 Pathogens.................................................................................................................................20 2.1.3 Oligonucleotides......................................................................................................................21 2.1.4 Chemicals................................................................................................................................21 2.1.5 Peptides....................................................................................................................................21 2.1.6 Antibiotics................................................................................................................................21 2.1.7 Media.......................................................................................................................................21 2.1.8 Antibodies................................................................................................................................22 2.1.9 Buffers and solutions ..............................................................................................................23 2.2 Methods............................................................................................................................................23 2.2.1 Maintenance and cultivation of Arabidopsis...........................................................................23 2.2.2 Arabidopsis seed sterilization..................................................................................................23 2.2.3 Maintenance of Pathogens.......................................................................................................24 2.2.4 Pathogen infection assays and quantification..........................................................................24 2.2.5 Bioassays to monitor PAMP responses....................................................................................24 2.2.5.1 ROS detection in seedlings.............................................................................................24 2.2.5.2 ROS detection in mature plants......................................................................................25 2.2.5.3 Ethylene measurement....................................................................................................25 2.2.5.4.Membrane depolarization...............................................................................................25 2.2.5.5 Arrest of seedling growth................................................................................................25 2.2.5.6 Analysis of Callose deposition........................................................................................26 2.2.6 Biochemical Molecular biological methods............................................................................26 2.2.6.1 In-gel MAP kinase assay.................................................................................................26 2.2.6.2 Total protein extraction and Immunoblot analysis..........................................................28 2.2.6.3 Total RNA extraction and Reverse-Transcription Polymerase Chain Reaction (RT-PCR)............................................................................................................................................28 2.2.6.4 Isolation of genomic DNA from Arabidopsis.................................................................28 2.2.6.5 Polymerase Chain Reaction (PCR).................................................................................29 3 Results.......................................................................................................................................................30 3.1.1 PAMP-triggered ROS production requiresAtRbohD important for immunity.............................30 3.1.2 Concluding remarks......................................................................................................................32 Supplementary figures...........................................................................................................33 3.2.1 Identification of mutants reduced in ROS production...................................................................35 3.2.2 Flg22-dependent responses inriomutants....................................................................................36 3.2.3 Concluding remarks......................................................................................................................38 Supplementary figures...........................................................................................................39 3.3.1 ROS production in response to flg22 is diminished in ethylene insensitive mutants...................41 3.3.2 Ethylene and ethylene signaling controlling FLS2 ......................................................................43 3.3.3 Concluding remarks......................................................................................................................46 Supplementary files...............................................................................................................48 4 Discussion.................................................................................................................................................51 4.1 PAMP-triggered oxidative burst contributes to plant immunity......................................................51 4.2 Molecular components of PAMP-triggered ROS.............................................................................52
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4.3 Components of ethylene signaling regulate ROS production..........................................................53 4.4 Final remarks and perspectives........................................................................................................55 References....................................................................................................................................................59 List of figures...............................................................................................................................................69 List of supplementary figures......................................................................................................................70 List of tables.................................................................................................................................................71 Acknowledgments........................................................................................................................................73 Erklärung......................................................................................................................................................75
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Abbreviations
:: fused to (in context of gene fusion constructs) % (v/v) volume percent % (w/v) weight/volume percent 3’ downstream region (of a gene or sequence) 5’ upstream region (of a gene or sequence) µ micro A adenine aa amino acid ABA abscisinic acide ACC 1-aminocyclopropane-1-carboxylic acid APS ammonium persulfate ATP adenosine trisphosphate At, A.th., Arabidopsis thaliana Arabidopsis Arabidopsis thaliana Arg arginine AVG amino-vinyl glycine avr avirulence °C degrees Celsius Ca2+calcium ions cfu colony formin units cDNA copy DNA CEBiP chitin oligosaccharide elicitor-binding protein Col-0 Arabidopsis thaliana ecotype Columbia-0 C-terminus carboxy terminus dH2O de-ionized water DMSO dimethyl sulfoxide DNA desoxy ribonucleic acid dNTPs desoxyribonucleotides dpi days post inoculation DTT dithiothreitol EDTA ethylene diamine tetra-acetate EFR EF-Tu receptor EMS ethyl methane sulfonate, or methane sulfonic acid ethyl ester ET ethylene ETI effector-triggered immunity flg flagellin FLS2 flagellin sensing receptor 2 G guanine h hour H2O2hydrogen peroxide HO.hydroxyl radical hpi hours post inoculation HR hypersensitive response HRP horse radish peroxidase JA jasmonic acid K kilo kb kilo base kD kilo Dalton l liter LRR leucine-rich repeats m milli
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M MAP4 MAPK MeOH min mRNA n NADPH NASC Nb Nt N-terminus OD Os O2 O2− P p35S PAGE PAMP PCR pH PRR Pfu PM PTI pv. RT-PCR RbohD RbohF RbohC RLK RLP ROS RNA rpm RT s SA SD SDS SEM Taq TBS TBS-T TEMED u V v w WT
molar (mol/l) mammalian microtubule-associated protein 4 mitogen activated protein kinase methanol minutes messenger RNA nano Nicotinamide adenine dinucleotide phosphate Nottingham Arabidopsis Stock Centre Nicotiana benthamiana Nicotiana tabacum amino terminus optical density Oryza sativa oxygen superoxide anion probability value promoter of Cauliflower mosaic virus promoter 35S polyacrylamide gel electrophoresis pathogen-associated molecular pattern polymerase chain reaction negative logarithm of proton concentration Pattern-recognition receptor Pyrococcus furiosus plasma membrane PAMP-triggered immunity pathovar reverse transcription-polymerase chain reaction Respiratory burst oxidase homologue D Respiratory burst oxidase homologue F Respiratory burst oxidase homologue C receptor-like kinase receptor-like protein reactive oxygen species ribonucleic acid rounds per minute room temperature seconds salicylic acid standard deviation sodium dodecyl sulphate standard error of the mean Thermophilus aquaticus tris buffered saline TBS with 0,5% Tween-20 N,N,N',N'-Tetramethylethylenediamine (enzymatic) unit volt volume weight wild-type
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Publications
Mersmann, S., Salomon, S., Vetter, M., and Robatzek, S. (2008) Selbst oder Nicht-Selbst– Pflanzliche Immunrezeptoren. BIOspektrum 14, 6, 593-596.
Goehre, V., Spallek, T., Haeweker, H., Mersmann, S., Mentzel, T., Boller, T., Torres, M., Mansfield, J.W., Robatzek, S. (2008) Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Current Biology, 18, 1824-1832.
Mersmann, S., Rietz, S., Chinchilla, D., and Robatzek, S. (2009) Ethylene signaling functions in NADPH oxidase RbohD mediated oxidative burst required for plant immunity. (submitted)
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Summary
Reactive oxygen species (ROS) are important molecules that are rapidly generated in response to abiotic and biotic stimuli and which regulate diverse physiological processes such as stomatal aperture and cell death. Plant immunity involves the detection of pathogen-associated molecular patterns (PAMPs) through cognate pattern recognition receptors (PRRs). Perception of PAMPs induces an extracellular oxidative burst, which requires the function of the NADPH oxidase AtRbohD. However, little is known about the regulation of PAMP-elicited ROS and its role in plant PAMP-triggered immunity. We investigated ROS production mediated by the FLS2 receptor kinase responsible for the detection of bacterial flagellin (flg22) in Arabidopsis to elucidate components of early flg22 signaling. Rboh proteins are assumed to predominantly mediate extracellular ROS production in plants. We observedAtbe rate limiting for flg22-elicited ROS production. Moreover,RbohD to bacterial multiplication monitored at early stages of infection with a disarmed but pathogenic Pseudomonas syringaeindicated ROS accumulation important for plant immunity.strain A forward genetic screen led to the isolation ofrio1 torio5 all of which were mutants, severely reduced in flg22-induced ROS production.Rio1,rio2andrio3carry mutations inFLS2and its co-receptorBAK1, respectively. The responsible gene mutations ofrio4 andrio5 remain to be identified in a subsequent mapping approach. A candidate gene approach revealed that flg22-stimulated oxidative burst was specifically inhibited in ethylene signaling mutants. Impaired ROS production inetr1andein2mutants could be partially rescued by chemical interference with ethylene accumulation. Notably, wounding partially complemented the ROS reduced phenotype. Furthermore, accumulation of FLS2 but not BAK1 was in part dependent on ethylene signaling. Bacterial multiplication at early time points was significantly enhanced in ethylene signaling mutants indicating the importance of PAMP-triggered ROS production in plant defence responses.
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Zusammenfassung
Reaktive Sauerstoff Spezies (ROS), deren Generierung durch abiotische und biotische Stimuli initiiert wird, sind für die Regulierung diverser physiologische Prozesse wichtige Moleküle, wie zum Beispiel der Öffnung von Stomata oder dem Zelltod. Die pflanzliche Immunität basiert auf der Erkennung von Pathogen assoziierten molekularen Mustern (PAMPs) durch Muster Erkennungs Rezeptoren (PRRs). Die Perzeption von PAMPs elizitiert einen extrazellulären oxidative Burst, der die Funktion der NADPH OxidaseAtRbohD erfordert. Bisher ist wenig über die Regulierung PAMP-induzierten ROS, sowie dessen Rolle in der planzlichen Immunität bekannt. Die Rezeptor Kinase FLS2 perzeptiert und bindet bakterielles Flagellin (flg22) und vermittelt dadurch die Produktion von ROS. In dieser Arbeit untersuchen wir die FLS2 induzierte ROS Generierung, um Komponenten, die diese Signaltransduktion vermitteln, zu finden. Die Produktion extrazellulären ROS wird hauptsächlich durch Rboh Oxidasen bewerkstellingt und zeigt die Wichtigkeit dieser Proteine. Unsere Untersuchung ergab, dassAtRbohD die limitierende Oxidase für den flg22-vermittelen oxidativen Burst ist. Überdies deuten unsere Ergebnisse darauf hin, dass ROS, welche durchAtRbohD generiert werden, entscheidend zur pflanzlichen Immunität beitragen, da die bakterielle Multiplikationsrate eines abgeschwächt pathogenensomanPsdoeu Stammes in frühen Zeitpunkten der Infektion signifikant erhöht ist. Mittels einer genetischen Untersuchung konnten wir die Mutantenrio1 bisrio5 isolieren, die alle eine Reduktion der ROS Produktion aufweisen. Weitere Untersuchungen ergaben, dassrio1, rio2undrio3Mutationen in entwederFLS2oder dessen KorezeptorBAK1tragen. Die Gene, die die Mutationen aufweisen, welche für die Phänotypen vonrio4 undrio5 sind, konnten verantwortlich bisher nicht identifiziert werden. Das deutet darauf hin, dassrio4undrio5 neue Komponenten der Signaltransduktion repräsentieren. Weitere genetische Verfahren sind erforderlich, um die Identität der betroffenen Gene zu analysieren. In einem weiteren genetischen Ansatz mit bisher bekannten Mutanten konnten solche, die Ethylen insensitiv sind, als reduziert in der flg22-vermittelten ROS Produktion gefunden werden. Die Verminderung der ROS Generierung inetr1undein2Mutanten konnte teilweise durch Ethylen-Biosynthese Inhibitoren aufgehoben werden. Interessanterweise wurde die Reduktion der ROS Generierung in verwundeten Blättern deretr1 undein2 Mutanten nicht beobachtet. Die Akkumulierung von FLS2 aber nicht von BAK1 war anteilig von der Ethylen Signaltransduktion abhängig. Die bakterielle Multiplikationsrate in frühen Zeitpunkten der Infektion war inetr1 und ein2 MutantenDas impliziert die Relevanz der PAMP-vermittelter ROS Produktion in der erhöht. pflanzlichen Immunität.
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